Human serum albumin binds and transports in the blood a myriad of small hydrophobic ligands such as bilirubin, tryptophan, fatty acids and a large number of commonly administered drugs (e.g., digitoxin, propranolol, furosemide, diazepam, amitriptyline, phenytoin, nafcillin, warfarin, tolbutamide, phenylbutazone and indomethacin). These tightly bound drugs (90 to 99+% bound) sometimes displace one and other or normal ligands in clincally important displacement reactions (which may be allosteric or competitive in nature). Despite the obvious bio-medical importance of human albumin: ligand interactions there has been little progress made towards determining the molecular details of metabolite and drug binding in the normal state or alterations which occur in various disease states. The complexity of the multiple ligand:albumin interactions dictates a requirement for either site specific probes or albumin dissection in order to determine the number, location and molecular characteristics of individual binding sites. In preliminary experiments we have used controlled limited proteolysis with pepsin at pH 3.7 in the presence of a protecting ligand. Fragments corresponding to residues 1-506, 1-455, 1-387, 1-308, 309-585, 49-308, 309-387 and 507-585 have been purified to varying degrees by affinity chromatography and conventional techniques. Three fragments without internal cleavages (1-387, 1-308, 49-308) possess the primary bilirubin binding site. Additional drug, tryptophan and fatty acid binding fragments will be rapidly prepared based on the results of novel screening procedure. Fragments will be purified and characterized with the aid of HPLC procedures. The number, location and affinities of the several binding sites will be determined by a uniquely modified (dual label) competitive protein binding technique. Radiochemical modification of specific amino acid residues will be undertaken (+/- ligands) in a manner not previously employed. These procedures are designed to retain native structure and to rapidly assess effects of ligand binding on the accessibility of reactive residues. Recently we have demonstrated that several peptic fragments prepared at low pH spontaneously reassociate under alkaline conditions for form albumin-like complexes. Similar modification in the presence and absence of reassociating fragments will allow us to determine the contact points.